An in situ protocol for mammalian cell mutagenesis assays, in which cells are fixed during the expression and selection phases, was developed. It allows for the calculation of the mutation frequency, the proportion of new mutations in a population of cells, and rather than the mutant fraction, the proportion of mutants in a population of cells. The mutant fraction can give misleading assessments of the mutagenic activity of chemicals when a large number of mutants grow more slowly than the rest of the population. This protocol permits the calculation of mutation rates and we anticipate that it will give a more accurate assessment of the mutagenic activity of chemicals than standard protocols.